PROJECT SUMMARY_____ __________________________________________ __ Traumatic brain injury (TBI) is a leading cause of mortality and long-term disability worldwide. Over 1.7 million Americans suffer a TBI annually and up to 2% of the population currently lives with the long-term neurological consequences of a previous TBI, placing a $76.5 billion annual economic burden on society. Preventative measures reduce injury incidence and/or severity, yet one-third of hospitalized TBI patients die from injuries that are secondary to the initial trauma. Cerebral edema is a life-threatening neurological complication that promotes elevated ICP and leads to clinical deterioration in the hours and days after a TBI. Unfortunately, neurosurgical approaches to control elevated ICP are limited and efficacious medical therapies to control cerebral edema are lacking, presenting a critical barrier to improving patient prognoses after TBI. The objective of this proposal is to test the overarching hypothesis that activation of neutrophilic toll-like receptor 4 (TLR4) promotes the formation of neutrophil extracellular traps (NETs) after TBI, initiating a detrimental immune cascade that culminates in neurological deterioration. Specific Aim 1 will test the hypothesis that TLR4 activation mediates NET formation after TBI. Proposed mechanistic studies will demonstrate a key regulatory role for activation of the TLR4 signaling pathway in the formation of NETs after TBI. Specific Aim 2 will test the hypothesis that peptidylarginine deiminase 4 (PAD4) promotes cerebral edema after TBI. Proposed mechanistic studies will use genetic and pharmacological approaches to implicate PAD4, a mediator of TLR4-induced histone hypercitrullination in human and mouse neutrophils, in NET formation and neurovascular injury after TBI. Specific Aim 3 will test the hypothesis that NETs trap macrophages in the brain after TBI. These studies will identify NETs as a novel mediator of brain macrophage infiltration/polarization after TBI. Proposed translational studies also will support the repurposing of recombinant human DNase (rhDNase), a safe, FDA-approved therapeutic in widespread clinical use for non- neurological diseases, allowing rapid clinical translation into TBI patients. Expected outcomes of the proposed research include the identification of NETs as a functional initiator of secondary neurological injury after TBI, establishing a framework for the development of innovative therapeutics to reduce neurovascular injury and improve patient outcomes after TBI. .